1. Technical Field
The present invention relates to analytical separation technology and more specifically towards capillary electrochromatography.
2. Background Art
Various capillary chromatographic separation techniques exist for analytical separation of various substances, compounds, and solutes. Some examples of capillary chromatographic separation methods include, but are not limited to, high-performance liquid chromatography (hereinafter “HPLC”), capillary gas chromatography, capillary electrochromatography (hereinafter “CEC”), and supercritical chromatography. Specifically, CEC is considered a rapidly growing technique in the area of analytical separations. CEC is a hybrid technique between HPLC and capillary zone electrophorens (CZE). The technique of CEC basically involves packing CZE capillaries with HPLC packing, applying a voltage across the packed capillary, and lining an organo-aqueous electrolyte solution. The applied voltage generates an electroosmotic flow (hereinafter “EOF”) across the capillary, wherein the differential partitioning and electrophoretic migration of solutes occur during transportation by the EOF resulting in CEC separation.
The current interest in CEC arises primarily from the enhanced separation efficiencies and peak capacities generated in the electrically driven CEC system over that of its conventional counterpart, the pressure driven HPLC system. Additionally, while CZE is applicable only to the separation of electrically charged analytes, CEC can separate both charged and neutral species. This constitutes a major advantage of CEC over CZE. As in HPLC, the separation in CEC is based upon the analyte's differential interactions between the mobile and stationary phases. The plug-like flow profile generated in CEC is responsible for the enhanced separation efficiencies. The use of CEC as an independent separation technique however, requires effective solution of a number of problems. One major problem lies in the area of column technology. The main focus here is to create CEC columns with stable controllable EDF that will allow fritless operation, and prevent bubble formation during a CEC run.
Sol-gel chemistry is an approach that is very applicable to CEC column technology. Sol-gel chemistry provides a general approach to prepare surface coatings on substrates of various dimensions and configurations. It allows for in situ creation of chemically bonded coatings on both the inner surface of capillaries and tubes, as well as on the outer surface of solid substrates of various shapes and sizes. Sol-gel chemistry is applicable for the preparation of both thin (<1 μm) and thick (>1 μm) coatings that possess high operational stability. Additionally, sol-gel chemistry has been successfully used as coatings for separation media located within various separation devices including, but not limited to, open tubular columns with surface-bonded octadecylsilane (hereinafter “ODS”) coatings in columns for use in open tubular capillary electrochromatography (hereinafter “OT-CEC”). The use of such sol-gel chemistry in column manufacture improves performance.
As for specific formats, an open tubular format represents a conceptually simple column design in CEC. In this format, a stable surface-bonded coating needs to be created on the inner walls of a capillary to provide efficient chromatographic separation and reliable electroosmotic flow. The OT-CEC capillaries more closely resemble those used in capillary zone electrophoresis (hereinafter “CZE”), therefore commercially available instruments designed for CZE can be used for CEC operations without requiring any instrumental modifications (e.g., pressurization capabilities exist). Furthermore, sample introduction is not limited to the biased electrophoretic mode as the pressurization levels required for hydrodynamic injections are readily achievable when open tubular columns are used. Open tubular columns do not require the use of retaining end-frits and packing materials, and therefore are practically free from bubble formation problems and other technical difficulties associated with the use of packed capillary columns.
OT-CEC columns have been used for various applications. For instance, Tsuda et al. demonstrated the separation of a series of hydrocarbons on ODS coated capillaries. For this, the 30-μm i.d. soda lime capillaries were first treated with sodium hydroxide, followed by the attachment of the stationary phase using silane chemistry. Bruin and co-workers (Bruin et al.) have used octadecylsilane coated 10–25 μm i.d. OT-CEC capillaries for the separation of a test mixture of polycyclic aromatic hydrocarbons (PAHs). Using applied voltages of 20 kV and a 1:1 50 mM phosphate/methanol mobile phase, plate height values of 1.2 μm were obtained for PAHs. Pesek and Matyska also reported the use of C18 bonded stationary phases within previously etched 50 μm i.d. fused-silica capillaries. In these studies, capillary modifications were performed using triethoxysilane, accompanied by a subsequent reaction with octadecene. Test mixtures of proteins and peptides and of tetracyclines were separated using a methanolic mobile phase. Yeung and co-workers (Pfeffer et al. and Garner et al.) added tetrabutylammonium (TBA) to the buffer solution and separated naphthalenesulfonic acid anions on OT-CEC capillaries. Tan and Remcho used a polymethacrylate coating within 25 μm i.d. capillaries for use in OT-CEC. Prepared through the polymerization of methacrylates, these OT-CEC columns generated efficiencies of up to 270,000 plates/m when separating a mixture of four benzoates.
Sawada et al. recently prepared coated open tubular columns for CEC using a copolymerization of N-tert-butylacrylamide (TBAAm) with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS). EOF, essential in CEC, can be generated as a result of the incorporation of the negatively charged AMPS into the chemical structure of the polymeric coating. Columns prepared by this in situ copolymerization technique proved successful for the separation of ketones, parabens, and PAHs. Guo et al. used sol-gel technology to cast a thin film on the inner fused-silica capillary surface for use in OT-CEC. By altering the percentage of octyl moieties, successful separation of a test mixture of five PAHs was achieved.
Despite the simplicity in design and user-friendliness in operation, open tubular columns are not as widely used as the packed capillary or monolithic columns in current CEC practice. This is partially attributed to slow solute diffusion in a liquid mobile phase and to a low sample capacity of the open tubular columns. To undergo interaction with the stationary phase, analytes must travel a significant distance (on a molecular scale) through the mobile phase. Thus, the column internal diameter must be small, the stationary phase film must be thick, and the column length must be great to render sufficient chromatographic interactions in the OT-CEC. To overcome these limitations a number of different approaches have been used in the preparation of open tubular columns.
One of the first published approaches to overcome the above mentioned limitations involved etching the capillary inner surface prior to affixing a chromatographically favorable stationary phase. (Hibi, et al., Jorgenson et al., Ishii et al., and Tsuda et al.). This etching process was primarily employed to increase the surface area of the smooth capillary inner walls, thereby providing favorable phase ratio values. Unfortunately, low retentions and sample capacities were inherent characteristics of columns prepared by this technique.
A second approach to the fabrication of open tubular columns involved first casting a siliceous sub-layer along the inner capillary surface. Again, as in the use of etched surfaces, this layer of silica is used to increase the capillary's inner surface area. Next, a monomeric stationary phase is chemically attached to this sublayer, thus creating a chromatographically favorable coated surface. However, the reproducibility of preparing homogeneous capillaries using this bi-modal technique is poor.
A third approach to the fabrication of open tubular columns involved the use of relatively thick, immobilized, polymeric films to enhance solute sorption. (Van Berkel et al., Folestad et al., van Berkel et al., Gohlin et al. (1991), Gohlin et al. (1993), Jorgenson et al., Ruan et al., Swart et al.). The creation of a cross-linked coating along the inner fused-silica capillary surface quickly gained interest due to the ease of column fabrication. Despite poor solute diffusion rates and stationary-phase bleeding, this technique was often employed for the preparation of open tubular columns for liquid-phase separations.
Accordingly, there is a need for an improved column and method of manufacturing the column thereof wherein increased performance of separation occurs through the use a prepared open tubular column coated with a specific sol-gel solution. Further, there is a need for controlling EOF in order to improve separation of electrically charged and/or neutral analytes by CEC.